Vascularization enhanced graft constructs

ABSTRACT

A tissue graft construct for use in repairing diseased or damaged tissues is provided. The tissue graft construct comprises a matrix composition selected from the group consisting of liver basement membrane and extracts and hydrolysates thereof, and processed collagen from vertebrate non-submucosal sources, added endothelial cells, and at least one additional preselected, exogenous population of cells which enhance the initiation of vessel-like structures in the grant. The preselected population of cells can be a population of non-keratinized or keratinized epithelial cells or a population of mesodermally derived cells selected from the group consisting of fibroblasts, smooth muscle cells, skeletal muscle cells, cardiac muscle cells, multi-potential progenitor cells, pericytes, osteogenic cells, and any other suitable cell type, preferably selected based on the tissue to be repaired. Methods for enhancing the vascularization in vivo of these tissue graft constructs and for preparing these graft constructs are also provided.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a divisional application of U.S. patent applicationSer. No. 10/428,355, filed May 2, 2003, which claims priority under 35U.S.C. §119(e) to U.S. Provisional Application No. 60/377,565, filed May2, 2002.

GOVERNMENT RIGHTS

This invention was made with government support under Grant No.GM60691-01 awarded by the National Institutes of Health. The governmenthas certain rights in the invention.

FIELD OF THE INVENTION

The preset invention relates to vascularization enhanced tissue graftsderived from a matrix composition and their use in repairing diseased ordamaged tissues. More particularly, this invention is directed tovascularization enhanced tissue grafts comprising a matrix compositionthat has been seeded with endothelial cells and at least one additionalpreselected, exogenous population of cells to enhance the repaircapabilities of the tissue graft constructs.

BACKGROUND AND SUMMARY OF THE INVENTION

The present invention is directed to a tissue graft construct comprisinga matrix composition seeded with endothelial cells and at least oneadditional preselected, exogenous cell population for use in the repairof damaged or diseased tissues. The matrix composition for use inaccordance with the present invention is selected from the groupconsisting of liver basement membrane and extracts and hydrolysatesthereof, and processed collagen from vertebrate non-submucosal sources.The matrix composition preferably comprises highly conserved collagens,glycoproteins, proteoglycans, and glycosaminoglycans. The matrixcomposition for use in this invention is derived from the tissue of awarm-blooded vertebrate.

The tissue graft constructs prepared in accordance with the presentinvention are substantially acellular matrices that provide a superiorcell growth substrate resembling the matrix environment found in vivo.The natural composition and configuration of the matrix compositionprovides a unique cell growth substrate that promotes the attachment andproliferation of cells in vitro and induces tissue remodeling when thegraft constructs are implanted in vivo.

As tissue graft materials, liver basement membrane and extracts andhydrolysates thereof, and processed collagen from vertebratenon-submucosal sources, induce the growth of endogenous tissues uponimplantation into a host (i.e., the graft materials induce remodeling).When used in such an application the tissue graft constructs appear notonly to serve as a matrix for the growth or regrowth of the tissuesreplaced by the graft constructs, but also to promote or to induce suchgrowth or regrowth of endogenous tissue. These graft materials can beused in an implantable sheet form or in injectable fluidized or gelforms for inducing the regrowth of endogenous tissues.

The present invention is directed to tissue graft constructs comprisinga matrix composition selected from the group consisting of liverbasement membrane and extracts and hydrolysates thereof, and processedcollagen from vertebrate non-submucosal sources, and further includingadded endothelial cells and at least one additional preselected,exogenous population of cells. The invention is also directed to methodsof enhancing the vascularization of a tissue graft construct in vivo.The vascularization enhanced tissue graft constructs are prepared byseeding the matrix composition in vitro with endothelial cells orendothelial cell precursors (e.g., progenitor cells or stem cells) andat least one additional preselected or predetermined cell type prior toimplanting or injecting the tissue graft construct into the host.

One embodiment provides a tissue graft construct for use in repairingdiseased or damaged tissues. The tissue graft construct comprises amatrix composition selected from the group consisting of liver basementmembrane and extracts and hydrolysates thereof, and processed collagenfrom vertebrate non-submucosal sources, added endothelial cells, and atleast one additional preselected, exogenous population of cells.

In another embodiment a vascularized tissue graft construct is providedfor use in repairing diseased or damaged tissues. The tissue graftconstruct comprises a matrix composition selected from the groupconsisting of liver basement membrane and extracts and hydrolysatesthereof, and processed collagen from vertebrate non-submucosal sources,added endothelial cells, and at least one additional preselected,exogenous population of cells wherein the endothelial cells have beencultured on the matrix composition for a time sufficient to form vesselsor vessel-like structures in vitro.

In another embodiment a method is provided for enhancing thevascularization in vivo of a tissue graft construct. The methodcomprises the steps of seeding in vitro a matrix composition selectedfrom the group consisting of liver basement membrane and extracts andhydrolysates thereof, and processed collagen from vertebratenon-submucosal sources, with a population of endothelial cells and atleast one additional preselected, exogenous population of cells to formthe graft construct, and implanting the graft construct into avertebrate at a site in need of repair.

In yet another embodiment a method is provided for enhancing thevascularization in vivo of a tissue graft construct. The methodcomprises the steps of seeding in vitro a matrix composition selectedfrom the group consisting of liver basement membrane and extracts andhydrolysates thereof, and processed collagen from vertebratenon-submucosal sources, with a population of endothelial cells and atleast one additional preselected, exogenous population of cells,culturing in vitro the endothelial cells for a time sufficient to inducethe formation of vessels or vessel-like structures or components, andimplanting the graft construct into a vertebrate in a site in need ofrepair.

In either of these method embodiments the matrix composition can beseeded with the additional preselected population of cells after thematrix composition is seeded with the endothelial cells, the matrixcomposition can be seeded with the additional preselected population ofcells before the matrix composition is seeded with the endothelialcells, or the matrix composition can be seeded with the endothelialcells and the additional preselected population of cells simultaneouslyor nearly simultaneously.

The endothelial cells can be cultured in vitro on the matrix compositionfor a time sufficient to induce the formation of vessels or vessel-likestructures, or the endothelial cells can be cultured on the matrixcomposition for a time sufficient to expand the endothelial cells (i.e.,allow the endothelial cells to divide at least one time) without formingvessels or vessel-like structures in vitro. Alternatively, the graftconstruct can be implanted without expanding the endothelial cells. Inany of these embodiments the additional preselected population of cellsmay or may not be expanded (i.e., allowed to progress through at leastone cell division cycle) prior to implanting the graft construct.

In still another embodiment a method is provided of preparing a tissuegraft construct for use in repairing diseased or damaged tissues. Themethod comprises the step of seeding in vitro a matrix compositionselected from the group consisting of liver basement membrane andextracts and hydrolysates thereof, and processed collagen fromvertebrate non-submucosal sources, with a population of endothelialcells, and at least one additional preselected, exogenous population ofcells to form the graft construct. The method can further comprise thestep of culturing the endothelial cells in vitro on the matrixcomposition for a time sufficient to induce the formation of vessels orvessel-like structures.

In any of these embodiments the at least one additional cell populationcan comprise a population of non-keratinized or keratinized epithelialcells or a population of mesodermally derived cells selected from thegroup consisting of fibroblasts, smooth muscle cells, skeletal musclecells, cardiac muscle cells, multi-potential progenitor cells (e.g.,stem cells), pericytes, and osteogenic cells. In various embodiments,the matrix composition can be seeded with endothelial cells and one ormore of these additional cell types (i.e., the matrix composition can beseeded with endothelial cells and one, two, three, etc. of theseadditional cell types).

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 provides flow charts depicting alternative preparations of thepresent graft constructs.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to a tissue graft construct comprisinga matrix composition selected from the group consisting of liverbasement membrane and extracts and hydrolysates thereof, and processedcollagen from vertebrate non-submucosal sources, and further includingadded endothelial cells and at least one additional preselected,exogenous population of cells. The matrix composition is seeded with theendothelial cells and the preselected, exogenous population(s) of cells,and is used to repair diseased or damaged tissues. In accordance withthe invention “damaged tissues” means tissues which are injured,lacerated, severed, or that have been surgically removed or areotherwise missing from the site in need of repair (e.g., congenitalabsence or deformity).

The matrix composition can be prepared from an extracellular matrixcomposition derived from liver basement membrane and extracts andhydrolysates thereof. However, the matrix composition can also beprepared from other engineered tissues to form, for example, an isolatedbasement membrane layer, or from a commercially available processedcollagen composition, or a purified collagen composition. Exemplary ofcommercially available processed collagen compositions that may be usedin accordance with the invention are MATRIGEL®, ALLODERM®, INTEGRA®,APPLIGRAF®, DERMAGRAFT®, and PERI-GUARD®. MATRIGEL® Basement MembraneMatrix (Becton Dickinson) is a tumor-derived basement membranecomposition which is a soluble basement membrane extract of theEngelbreth-Holm-Swarm tumor, gelled to form a reconstituted basementmembrane. ALLODERM® (Life Cell, Inc.) is a composition from cadaverdermis that has been processed to remove cells. INTEGRA® (Integra LifeSciences) is an acellular dermal composition of bovine collagen andchondroitin sulfate. APPLIGRAF® (Novartis) is a synthetic polylacticacid-containing composition that has been seeded with human fibroblastsand other cellular and non-cellular components, and DERMAGRAFT® is anallogenic dermal graft of human fibroblasts on a Vicryl mesh backbone.PERI-GUARD® (Bio-Vascular, Inc.) is a composition prepared from bovinepericardium which is chemically cross-linked. Purified and processedcollagen can also be produced by techniques known in the art. (See, forexample, U.S. Pat. Nos. 6,127,143, 5,814,328, 5,108,424, and 4,883,864.)

The endothelial cells for use in accordance with the invention can bederived from any type of endothelial cell population includingmacrovascular, microvascular, arterial, and venous endothelial cells.Either mature endothelial cells (e.g., harvested from an organ or ablood vessel) or endothelial cell precursors (e.g., progenitor cells orstem cells) can be used in accordance with the invention. Additionally,the endothelial cells can be harvested from a young or an old animal,but endothelial cells harvested from a young animal are preferred.

In one embodiment the additional preselected, exogenous population(s) ofcells can comprise a population of non-keratinized or keratinizedepithelial cells or a population of mesodermally derived cells selectedfrom the group consisting of fibroblasts, smooth muscle cells, skeletalmuscle cells, cardiac muscle cells, multi-potential progenitor cells,pericytes, osteogenic cells, or any other suitable cell type.

The additional preselected, exogenous population of cells, which iscombined with the matrix composition and the endothelial cells, can beselected based on the cell type of the intended tissue to be repaired.For example, if skin is to be repaired, the preselected, exogenouspopulation of cells can be non-keratinized epithelial cells or ifcardiac tissue is to be repaired, the preselected, exogenous populationof cells can be cardiac muscle cells. In another embodiment the matrixcomposition is seeded with autogenous cells isolated from the patient tobe treated.

In one embodiment, the at least one additional preselected population ofcells to be combined with the matrix composition and the endothelialcells includes smooth muscle cells and/or progenitor cells capable ofdifferentiating into smooth muscle cells. Advantageously, the smoothmuscle cells and/or smooth muscle progenitor cells can promote, alongwith the endothelial cells, the formation of vessels or vessel-likestructures in the graft construct. In another embodiment, additionalcell types can be added along with endothelial cells, smooth musclecells, and/or smooth muscle cell progenitor cells.

In still another embodiment the at least one additional preselected,exogenous population of cells comprises a population of multi-potentialprogenitor cells. The matrix composition can induce the differentiationof these multi-potential progenitor cells into cells that assist in therepair of damaged tissues. Advantageously, the matrix composition seededwith a population of endothelial cells and a population ofmulti-potential progenitor cells can be implanted into a variety ofdifferent in vivo locations and the progenitor cells will differentiateinto the appropriate cell type for the specific environment. Forexample, implantation of a composition comprising endothelial cells andmulti-potential progenitor cells at the site of a tendon or a ligamentwill result in the graft construct remodeling into a tendon or aligament.

The combination of the matrix composition, endothelial cells, and anadditional preselected, exogenous population of cells provides a tissuegraft construct that shows surprisingly enhanced vascularization invitro and/or in vivo leading to improved wound healing capabilities andbetter restoration of tissue function compared to the use of either thematrix composition alone, in combination with endothelial cells alone,or in combination with cell types other than endothelial cells astherapeutic agents.

In various embodiments, the matrix composition can be seeded with theadditional preselected population of cells after the matrix compositionis seeded with the endothelial cells, the matrix composition can beseeded with the additional preselected population of cells before thematrix composition is seeded with the endothelial cells, or the matrixcomposition can be seeded with the endothelial cells and the additionalpreselected population of cells simultaneously or nearly simultaneously(see FIG. 1 for various exemplary embodiments).

In one such embodiment, the matrix composition can be seeded withendothelial cells and the endothelial cells can be cultured on thematrix composition prior to the implantation of the construct into theaffected region for a time sufficient to induce the formation of vesselsor vessel-like structures. The matrix composition can be seeded with theat least one additional preselected, exogenous population of cells afterthe matrix composition is seeded with the endothelial cells and at anytime up to just prior to implantation of the graft construct in vivo.Accordingly, depending on the time allowed for culturing the preselectedpopulation of cells on the matrix composition prior to implantation ofthe graft construct, the additional preselected population of cells mayor may not be expanded (i.e., allowed to progress through at least onecell division cycle) prior to implantation of the graft construct intothe affected region.

Alternatively, the matrix composition can be seeded with the at leastone additional preselected, exogenous population of cells after thematrix composition is seeded with the endothelial cells, and theendothelial cells can be cultured on the matrix composition to expandthe endothelial cells without inducing the formation of vessels orvessel-like structures or components prior to implantation of the graft.In this embodiment, depending on the time allowed for culturing thepreselected population of cells on the matrix composition prior toimplantation of the graft construct, the additional preselectedpopulation of cells may or may not be expanded prior to implantation ofthe graft construct into the affected region.

In another embodiment, the matrix composition can be seeded with the atleast one additional preselected, exogenous population of cells afterthe matrix composition is seeded with the endothelial cells and thematrix composition can be implanted soon thereafter without expansion ofeither the endothelial cells or the additional preselected, exogenouspopulation of cells.

In an alternate embodiment, the matrix composition can be seeded withthe additional preselected, exogenous population of cells and thepreselected population of cells can be cultured on the matrixcomposition to expand (i.e., allow the cells to divide at least onetime) the preselected cell population prior to implantation of the graftconstruct. In this embodiment, the matrix composition can be seeded withthe endothelial cells after the matrix composition is seeded with thepreselected population of cells and at any time up to just prior toimplantation of the graft in vivo. Accordingly, depending on the timeallowed for culturing the endothelial cells on the matrix compositionprior to implantation of the graft, the endothelial cells may or may notbe expanded prior to implantation of the graft construct into theaffected region. If the endothelial cells are expanded, the expansion ofthe endothelial cells may or may not include the formation of vessels orvessel-like structures.

In another embodiment, the matrix composition can be seeded with theendothelial cells after the matrix composition is seeded with the atleast one additional preselected, exogenous population of cells and thegraft can be implanted soon thereafter without expansion of either theendothelial cells or the additional preselected, exogenous population ofcells.

In yet another embodiment, the matrix composition can be seededsimultaneously or nearly simultaneously with the endothelial cells andthe additional preselected, exogenous population of cells. In thisembodiment, the endothelial cells and the additional preselected,exogenous population of cells can be cultured on the matrix compositionto expand the two cell populations or the graft can be implanted withoutexpansion of the two cell populations. If the endothelial cells areexpanded, the expansion of the endothelial cells may or may not includethe formation of vessels or vessel-like structures.

A matrix composition selected from the group consisting of liverbasement membrane and extracts and hydrolysates thereof, and processedcollagen from vertebrate non-submucosal sources, advantageously providesa physiological environment that supports the proliferation anddifferentiation of cells cultured in vitro on the matrix composition.Thus, cells can be seeded onto the matrix composition and can becultured using standard cell culture techniques, as described below,known to those of ordinary skill in the art, to produce tissue graftsfor implantation into a host in need thereof.

The ability of a matrix composition selected from the group consistingof liver basement membrane and extracts and hydrolysates thereof, andprocessed collagen from vertebrate non-submucosal sources, to provide asubstrate that supports the growth of cells provides the opportunity toexpand the population of endothelial cells and/or the additionalpreselected, exogenous population of cells prior to implantation into ahost. If endothelial cells are expanded, such expansion can result inthe formation of vessels or vessel-like structures (i.e., potentialvascularization of the graft construct in vitro) prior to implantationimproving the wound healing capabilities of the graft upon implantationof the graft construct. The formation of vessels or vessel-likestructures prior to implantation of the graft construct or,alternatively, the expansion of endothelial cells prior to implantationof the graft construct improves the wound healing capabilities of thegraft upon implantation such as by promoting differentiation andmigration of cells growing on the surface of the graft construct and bypromoting proliferation of cells within the graft construct.

In embodiments where the added endothelial cells, and the additionalpreselected, exogenous population of cells are cultured on the matrixcomposition prior to implantation, the cells are cultured on the matrixcomposition under conditions conducive to cell growth. The culturedcells can be in either direct or indirect contact (e.g., fluidcommunication) with the matrix composition. Conditions conducive to cellgrowth are environmental conditions, such as sterile technique,temperature (e.g., about 37° C.) and nutrient supply, that areconsidered optimal for cell growth under currently accepted proceduresfor tissue and cell culture. Although optimum culture conditions dependon the particular cell type, cell growth conditions are generally wellknown in the art.

Matrix compositions in accordance with the invention can be used in avariety of forms as a graft material and to culture endothelial cellsand other cell types in vitro prior to implantation of the graftconstruct. These forms include a sheet-like configuration, a gel form, afluidized composition (e.g., by comminuting or digesting the tissue),and an extract for addition to art-recognized cell/tissue culture media.The matrix composition or component(s) thereof can provide a surface forcell adhesion and/or can induce cell differentiation and/orproliferation. The matrix composition is preferably sterilized prior touse in tissue/cell culture applications, however, nonsterilecompositions can be used if antibiotics are included in the cell culturemedia.

In one embodiment cells are seeded directly onto sheets of liverbasement membrane tissue under conditions conducive to cellproliferation for culture of the cells prior to implantation of thegraft construct. The porous nature of this tissue allows diffusion ofcell nutrients throughout the matrix. Thus, cells can be seeded onto andcultured on either side of the liver basement membrane.

The endothelial cells and/or the additional preselected, exogenouspopulation of cells seeded onto the matrix composition for culture priorto implantation of the graft construct can be grown in the presence ofnutrients, including minerals, amino acids, sugars, peptides, proteins,or glycoproteins, such as laminin and fibronectin, and/or growth factorssuch as epidermal growth factor, vascular endothelial cell-derivedgrowth factor, platelet-derived growth factor, platelet-derived growthfactor-like molecules, transforming growth factor β, fibroblast growthfactor, or another serum growth factor. In one embodiment fluidized orpowder forms of the matrix composition can be used to supplementstandard cell culture media to enhance the capacity for sustaining andinducing the proliferation of the cells in vitro and to induceremodeling in vivo. The cells can be grown on the matrix composition inthe presence of commercially available cell culture liquid media (eitherserum-containing or serum-free).

In one embodiment, the at least one additional preselected population ofcells to be combined with the matrix composition and the endothelialcells can be smooth muscle cells and/or progenitor cells capable ofdifferentiating into smooth muscle cells to promote, along with theendothelial cells, the formation of vessels or vessel-like structures inthe graft construct. It is known that treatment of smooth muscle cellswith a heparinase can induce a phenotypic change characteristic ofproliferating cells. Accordingly, in embodiments where the matrixcomposition is seeded with endothelial cells and at least onepreselected, exogenous population of cells including a smooth musclecell population and/or a smooth muscle cell progenitor cell population aheparinase can be included in the cell culture medium. For example, 4units/ml of a heparinase from Flavobaterium heparinum can be included inthe culture medium for a short interval (e.g., 6 hours) or can bepresent continually in the culture medium.

It is also known that smooth muscle cells that are seeded on a substrateas a subconfluent monolayer of cells undergo a phenotypic changeassociated with the capacity to divide. The phenotypic change isinhibited if the smooth muscle cells are co-cultured with a confluentmonolayer of endothelial cells. Accordingly, in embodiments where thematrix composition is seeded with endothelial cells and at least onepreselected, exogenous population of cells including a smooth musclecell population and/or a smooth muscle cell progenitor cell populationthe added endothelial cells can be seeded onto the matrix composition sothat the cells attach to the matrix composition as a subconfluentmonolayer of cells. In another embodiment the endothelial cells, smoothmuscle cells, and/or smooth muscle progenitor cells can be seeded ontothe matrix composition so that the cells attach to the matrixcomposition as subconfluent monolayers of cells.

In one embodiment, the claimed compositions comprising the matrixcomposition, added endothelial cells, and an additional preselected,exogenous population of cells can be encapsulated in a biocompatiblematrix for implantation into a host. The encapsulating matrix can beconfigured to allow the diffusion of nutrients to the encapsulated cellswhile allowing the products of the encapsulated cells to diffuse fromthe encapsulated cells to the host cells. Suitable biocompatiblepolymers for encapsulating living cells are known to those skilled inthe art. For example a polylysine/alginate encapsulation process hasbeen previously described by F. Lim and A. Sun (Science, Vol. 210, pp.908-910). Indeed, liver basement membrane itself could be usedadvantageously to encapsulate the cells on a matrix in accordance withthis invention for implantation as an artificial organ.

In one embodiment, a method is provided for enhancing thevascularization in vivo of a tissue graft construct. The methodcomprises the steps of seeding in vitro a matrix composition selectedfrom the group consisting of liver basement membrane and extracts andhydrolysates thereof, and processed collagen from vertebratenon-submucosal sources, with a population of endothelial cells and atleast one additional preselected, exogenous population of cells to formthe graft construct, and implanting the graft construct into avertebrate at a site in need of repair. In one embodiment of thismethod, the matrix composition can be seeded with endothelial cells andthe endothelial cells can be cultured on the matrix composition prior tothe implantation of the construct into the affected region for a timesufficient to induce the formation of vessels or vessel-like structures.The matrix composition can be seeded with the at least one additionalpreselected, exogenous population of cells after the graft is seededwith the endothelial cells and at any time up to just prior toimplantation of the graft in vivo. Accordingly, depending on the timeallowed for culturing the preselected population of cells on the matrixcomposition prior to implantation of the graft construct, the additionalpreselected population of cells may or may not be expanded prior toimplantation of the graft construct into the affected region.

Alternatively, the matrix composition can be seeded with the at leastone additional preselected, exogenous population of cells after thematrix composition is seeded with the endothelial cells, and theendothelial cells can be cultured on the matrix composition to expandthe endothelial cells without inducing the formation of vessels orvessel-like structures prior to implantation of the graft. In thisembodiment, depending on the time allowed for culturing the endothelialcells on the matrix composition prior to implantation of the graftconstruct, the additional preselected population of cells may or may notbe expanded prior to implantation of the graft construct into theaffected region.

In another embodiment, the matrix composition can be seeded with the atleast one additional preselected, exogenous population of cells afterthe matrix composition is seeded with the endothelial cells and thegraft can be implanted soon thereafter without expansion of either theendothelial cells or the additional preselected, exogenous population ofcells.

In an alternate embodiment of this method, the matrix composition can beseeded with the additional preselected, exogenous population of cellsand the preselected population of cells can be cultured on the matrixcomposition to expand the preselected cell population prior toimplantation of the graft construct. In this embodiment, the matrixcomposition can be seeded with the endothelial cells after the matrixcomposition is seeded with the preselected population of cells and atany time up to just prior to implantation of the graft in vivo.Accordingly, depending on the time allowed for expansion of theendothelial cells by culturing the cells on the matrix composition priorto implantation of the graft, the endothelial cells may or may not beexpanded prior to implantation of the graft construct into the affectedregion. If the endothelial cells are expanded, the expansion of theendothelial cells may or may not include the formation of vessels orvessel-like structures.

In another embodiment, the matrix composition can be seeded with theendothelial cells after the matrix composition is seeded with the atleast one additional preselected, exogenous population of cells and thegraft can be implanted soon thereafter without expansion of either theendothelial cells or the additional preselected, exogenous population ofcells.

In yet another embodiment, the matrix composition can be seededsimultaneously or nearly simultaneously with the endothelial cells andthe additional preselected, exogenous population of cells. In thisembodiment, the endothelial cells and the additional preselected,exogenous population of cells can be cultured on the matrix compositionto expand the two cell populations or the graft can be implanted withoutexpansion of the two cell populations. If the endothelial cells areexpanded, the expansion of the endothelial cells may or may not includethe formation of vessels or vessel-like structures.

A vascularized tissue graft construct for use in repairing diseased ordamaged tissues is also provided in accordance with the invention. Thevascularized graft construct comprises a matrix composition selectedfrom the group consisting of liver basement membrane and extracts andhydrolysates thereof, and processed collagen from vertebratenon-submucosal sources, and further includes added endothelial cells,and at least one additional preselected, exogenous population of cellswherein the endothelial cells have been cultured on the matrixcomposition for a time sufficient to form vessels or vessel-likestructures in vitro.

In another embodiment, a method is provided for enhancing thevascularization in vivo of a tissue graft construct. The methodcomprises the steps of seeding a matrix composition selected from thegroup consisting of liver basement membrane and extracts andhydrolysates thereof, and processed collagen from vertebratenon-submucosal sources, in vitro with a population of endothelial cellsand at least one additional preselected, exogenous population of cells,culturing in vitro the endothelial cells and the additional cellpopulation on the matrix composition for a time sufficient to induce theformation of vessels or vessel-like structures, and implanting the graftconstruct into a vertebrate in a site in need of repair.

Matrix compositions in accordance with the invention can be seeded withinitially small cell populations that can be expanded in vitro prior toimplantation. Advantageously, seeding with endothelial cells can inducevascularization of the grafts in vitro upon culturing the endothelialcells in vitro on the matrix composition. The matrix composition can befurther seeded with smooth muscle cells or smooth muscle cell progenitorcells or another cell type, such as fibroblasts, to promotevascularization.

In this embodiment, the matrix composition is seeded with endothelialcells and the endothelial cells are cultured on the matrix compositionprior to the implantation of the construct into the affected region fora time sufficient to induce the formation of vessels or vessel-likestructures. The matrix composition can be seeded with the at least oneadditional preselected, exogenous population of cells after the matrixcomposition is seeded with the endothelial cells and at any time up tojust prior to implantation of the graft in vivo. Accordingly, dependingon the time allowed for culturing the preselected population of cells onthe matrix composition prior to implantation of the graft, theadditional preselected population of cells may or may not be expandedprior to implantation of the graft construct into the affected region.

In an alternate embodiment, the matrix composition can be seeded withthe additional preselected, exogenous population of cells and thepreselected population of cells can be cultured on the matrixcomposition to expand the preselected cell population prior toimplantation of the graft construct. In this embodiment, the matrixcomposition is seeded with endothelial cells after the matrixcomposition is seeded with the preselected population of cells. In thisembodiment, the endothelial cells are cultured on the matrix compositionfor a time sufficient to allow for expansion of the endothelial cells toform vessel or vessel-like structures prior to implantation of the graftconstruct into the affected region.

In another embodiment, the matrix composition can be seeded with theendothelial cells and the additional preselected, exogenous populationof cells simultaneously or nearly simultaneously. In this embodiment,the additional preselected, exogenous population of cells and theendothelial cells are cultured on the matrix composition to expand thetwo cell populations prior to implantation of the graft.

A method of preparing a tissue graft construct for use in repairingdiseased or damaged tissues is also provided. The method comprises thestep of seeding in vitro a matrix composition selected from the groupconsisting of liver basement membrane and extracts and hydrolysatesthereof, and processed collagen from vertebrate non-submucosal sources,with a population of endothelial cells, and at least one additionalpreselected, exogenous population of cells to form the graft construct.The method can further comprise the step of culturing the endothelialcells on the matrix composition for a time sufficient to induce theformation of vessels or vessel-like structures prior to the implantationof the graft construct into the affected region.

The matrix composition can be made from liver basement membrane (LBM)prepared by separating the LBM from the natively associated cellularcomponents of liver tissue of a vertebrate. The preparative techniquesdescribed below provide an extracellular matrix composition consistingessentially of LBM substantially free of any cellular components.

Basement membrane for use in the matrix composition in accordance withthe invention is typically prepared from liver tissue harvested fromanimals raised for meat production, including, for example, pigs, cattleand sheep or other vertebrates. Thus, there is an inexpensive commercialsource of liver tissue for use in preparation of the compositions usedin accordance with the present invention. The LBM composition does notinduce an adverse host immune response when the composition is used inthe delivery systems of the present invention.

To prepare the acellular LBM composition, the liver tissue is treatedwith a cell dissociation solution for a period of time sufficient torelease the cellular components of the liver tissue from theextracellular components without substantial disruption of theextracellular components, and the cellular components are separated fromthe extracellular components. Typically the cell dissociation solutioncomprises a chaotropic agent or an enzyme or both.

The first step in preparing LBM is to slice a segment of liver tissueinto pieces (e.g., strips or sheets) to increase the surfacearea-to-volume ratio of the liver tissue. The liver tissue may be slicedinto a series of sheets each having a thickness of about 50 to about 500microns, more preferably about 250 to about 300 microns. Freshlyharvested liver tissue can be sliced using a standard meat slicer, orthe tissue can be frozen and sliced with a cryomicrotone. The thinpieces of liver tissue are then treated with a solution that releasescomponent liver cells from the associated extracellular basementmembrane matrix.

The liver tissue can be treated with a solution comprising an enzyme,for example, a protease, such as trypsin or pepsin. Because of thecollagenous structure of the LBM and the desire to minimize degradationof the membrane structure during cell dissociation, collagen specificenzyme activity should be minimized in the enzyme solutions used forcell-dissociation. In addition, the liver tissue is typically alsotreated with a calcium chelating agent or chaotropic agent (e.g., TritonX-100). Thus, the liver tissue is treated by suspending slices or stripsof the tissue in a cell-dissociation solution containing enzyme(s) andchaotropic agent(s). However, cell dissociation can also be conductedusing a calcium chelating agent or chaotropic agent in the absence of anenzymatic treatment of the tissue.

Cell dissociation can be carried out by suspending, with agitation,liver tissue slices in a solution containing about 0.05 to about 2%,more typically about 0.1 to about 1% by weight of protease, optionallycontaining a chaotropic agent or a calcium chelating agent in an amounteffective to optimize release and separation of cells from the basementmembrane without substantial degradation of the membrane matrix.

After contacting the liver tissue with the cell-dissociation solutionfor a sufficient time to release all cells from the matrix, theresulting LBM is rinsed one or more times with saline and optionallystored in a frozen hydrated state or a partially dehydrated state untilused. Cell dissociation may require several treatments to releasesubstantially all cells from the basement membrane. The resulting LBMpreparation can be further treated to remove or inhibit any residualenzyme activity. For example, the resulting basement membrane can beheated or treated with one or more protease inhibitors.

In another embodiment, LBM can be prepared as follows. Freshly harvestedliver tissue can be sliced using a standard meat slicer into a series ofsheets each having a thickness of about 50 to about 2000 microns, or thetissue can be frozen and sliced with a meat slicer or cryomicrotone. Theliver tissue is then rinsed one or more times, such as with deionizedwater, saline, or a buffered solution and optionally stored in a frozenhydrated state or a partially dehydrated state until used. For example,the liver sheets or strips could be rinsed three times for 30 minuteseach with deionized water, saline, or a buffer. The rinse solution canthen be strained from the liver slices, for example, using a sieve, andeach liver slice can be massaged on a screen or ultrasound can be usedto hasten lysis of hepatocytes and to mechanically dissociatehepatocytes and hepatocyte cell fragments from the liver basementmembrane.

The thin slices of liver tissue are then contacted with a solutioncontaining a protease, such as trypsin, that releases liver cells andother components from the associated extracellular basement membranematrix. Because of the collagenous structure of the liver basementmembrane and the desire to minimize degradation of the membranestructure during cell dissociation, collagen specific enzyme activityshould be minimized in the enzyme solutions used in the proteasedigestion step. The liver tissue is typically also contacted with acalcium chelating agent such as EDTA concurrently with the proteasetreatment.

In one preferred embodiment the protease digestion step is carried outby contacting liver tissue slices with a solution, optionally withagitation, containing 0.02% of trypsin by weight and containing EDTA ata concentration of about 0.05% by weight. The protease digestion step ispreferably carried out with heating, typically at about 37° C. Therinsing and mechanical dissociation steps described above may berepeated after the protease digestion step.

The liver slices are then contacted with a solution containing anon-denaturing detergent. This step is preferably carried out at roomtemperature, and optionally with agitation. The non-denaturing detergentis preferably 3% Triton X-100. The rinsing steps described above arerepeated after contacting the liver slices with the non-denaturingdetergent to remove most, if not all, of the non-denaturing detergent.The mechanical dissociation steps may be repeated as needed.

After treatment with the non-denaturing detergent, the liver slices arecontacted with a solution containing a denaturing detergent. This stepis preferably carried out at room temperature and optionally withagitation. The denaturing detergent is preferably 4% deoxycholate. TheLBM is then thoroughly rinsed as described above to remove as muchresidual detergent as possible and the LBM can be stored (e.g., indeionized water at 4° C.) until further use or can be used immediatelyfollowing the purification procedure.

After preparation, LBM can be fluidized in a manner similar to thepreparation of fluidized submucosa, as described in U.S. Pat. No.5,275,826 the disclosure of which is expressly incorporated herein byreference. LBM is comminuted by tearing, cutting, grinding, shearing andthe like. Grinding the liver basement membrane in a frozen orfreeze-dried state is preferred although good results can be obtained aswell by subjecting a suspension of liver basement membrane to treatmentin a high speed (high shear) blender and dewatering, if necessary, bycentrifuging and decanting excess water. Additionally, the comminutedfluidized tissue can be solubilized by enzymatic digestion with aprotease, for example a collagenase and or other appropriate enzyme,such as glycanase, or other enzyme that disrupts the matrix structuralcomponents, for a period of time sufficient to solubilize the tissue andform a substantially homogeneous solution.

The viscosity of fluidized LBM for use in transdermal or transmucosaldrug delivery in accordance with this invention can be manipulated bycontrolling the concentration of the LBM component and the degree ofhydration. The viscosity can be adjusted to a range of about 2 to about300,000 cps at 25° C. Higher viscosity formulations, for example, gels,can be prepared from the LBM digest solutions by dialyzing the digestedmaterial and then adjusting the pH of such solutions to about 5.0 toabout 9.0.

LBM can also be prepared in the form of an extract. Briefly, LBM can besuspended in an extraction buffer with agitation for about 24 hours at4° C. The extraction mixture can be centrifuged at 12,000×g for about 30minutes at 4° C. and the supernatant collected. The insoluble materialcan then be washed with the extraction buffer and the centrifugationstep repeated and the wash combined with the original supernatant. Thesupernatant can be dialyzed (MWCO about 3500) extensively againstdeionized water and the dialyzate centrifuged at 12,000×g. Thesupernatant can be used immediately or lyophilized for storage.

Powder forms of LBM can also be used in accordance with the invention.Powder forms are prepared by pulverizing the tissue under liquidnitrogen to produce particles ranging in size from 0.1 to 1 mm². Theparticulate composition is then lyophilized overnight and sterilized toform a solid substantially anhydrous particulate composite.Alternatively, a powder form of LBM can be formed from fluidized LBM bydrying the suspensions or solutions of comminuted LBM. LBM in powderform or in fluidized form or in the form of a gel or an extract can beused to culture endothelial cells and the preselected cell population invitro prior to implantation of the graft construct in accordance withthe invention.

MATRIGEL®, ALLODERM®, and INTEGRA®, DERMAGRAFT®, PERI-GUARD®, andAPPLIGRAF® are commercially available processed collagen compositions.Purified and processed collagen compositions can also be prepared byprotocols known in the art. For example, see U.S. Pat. Nos. 6,127,143,5,814,328, 5,108,424, and 4,883,864 incorporated herein by reference.

EXAMPLE 1 Preparation of Liver Basement Membrane Compositions

2 mM EDTA Buffered Chaotropic Solution Used in the Experiment

140 mM NaCl 5 mM KCl 0.8 mM MgSO₄ 0.4 mM KH₂HPO₄ 2 mM EDTA 25 mM NaHCO₃Procedure:

Preparation of Liver Slices:

Liver frozen at −70° C. was sliced with a cryomicrotone to a thicknessof about 50μ. The slices of liver tissue were then subjected toenzymatic treatment (0.1% trypsin) with the chaotropic solution (2 mMEDTA) described above.

Liver slices were placed in five 50 ml tubes, each of which contained 25ml of the buffered enzyme treatment solution. The liver tissue wasincubated at 37° C. in water bath with gentle shaking for 1 hour. Theliver slices were washed twice with PBS with agitation/shaking for 1hour at room temperature. The above enzymatic treatment steps wererepeated three times. Frozen tissue was sliced into 1 cm cubes,pulverized under liquid nitrogen with an industrial blender to particlesless than 2 mm2 and stored at −80° C. prior to use.

Preparation of Extracts of Liver Basement Membrane7

Extraction buffers used for these studies included 4 M guanidine and 2Murea each prepared in 50 mM Tris-HCl, pH 7.4. The powder form of liverbasement membrane was suspended in the relevant extraction buffer (25%w/v) containing phenylmethyl sulphonyl fluoride, N-ethylmaleimide, andbenzamidine (protease inhibitors) each at 1 mM and vigorously stirredfor 24 hours at 4° C. The extraction mixture was then centrifuged at12,000×g for 30 minutes at 4° C. and the supernatant collected. Theinsoluble material was washed briefly in the extraction buffer,centrifuged, and the wash combined with the original supernatant. Thesupernatant was dialyzed extensively in Spectrapor tubing (MWCO 3500,Spectrum Medical Industries, Los Angeles, Calif.) against 30 volumes ofdeionized water (9 changes over 72 hours). The dialysate was centrifugedat 12,000×g to remove any insoluble material and the supernatant wasused immediately or lyophilized for storage.

Preparation of Fluidized Liver Basement Membrane

Partial digestion of the pulverized material described above wasperformed by adding 5 g of powdered tissue to a 100 ml solutioncontaining 0.1% pepsin in 0.5 M acetic acid and digesting for 72 hoursat 4° C. Following partial digestion, the suspension was centrifuged at12,000 rpm for 20 minutes at 4° C. and the insoluble pellet discarded.The supernatant was dialyzed against several changes of 0.01 M aceticacid at 4° C. (MWCO 3500). The solution was sterilized by addingchloroform (5 ml chloroform to 900 ml of 0.01 M acetic acid) to the LBMhydrolysate. Dialysis of the LBM was continued with two additionalchanges of sterile 0.01 M acetic acid to eliminate the chloroform. Thecontents of the dialysis bag were then transferred aseptically to asterile container. The resultant fluidized composition was stored at 4°C.

Preparation of Liver Basement Membrane Gel Compositions

To prepare the gel form of LBM, 8 mls of fluidized LBM was mixed with1.2 ml 10×PBS buffer (10× phosphate buffered saline containing 5 mg/Lphenol red); 0.04 N HCl (approx 1.6 ml) was added to adjust the pH tobetween 6.6 and 7.4 and then 0.05 N NaOH (approx. 1.2 ml) was added toshift the pH to >8. The final volume was adjusted to 12 ml with water.

EXAMPLE 2 Growth of Endothelial Cells

Liver basement membrane is prepared as described above. Followingsterilization via various techniques (gamma irradiation, peracetic acid,etc.), the tissue is clamped within a polypropylene frame to create aflat surface area (50 mm²) for cell growth. The frame is submerged intissue culture medium to allow access of medium nutrients to bothsurfaces of the liver basement membrane. Endothelial cells and smoothmuscle cells are seeded (at 3×10⁴ cells/tissue section) on the liverbasement membrane and then placed in a 5% CO₂, 95% air incubator at 37°C. Following various periods of time, the seeded liver basement membraneis fixed in 10% neutral buffered formalin, embedded in paraffin, andsectioned (6 um). Various histological and immunohistochemical stainingprocedures are done to determine the cell growth characteristics.Vessels or vessel-like structures are observed using these procedures.

1. A method for enhancing the vascularization in vivo of a tissue graftconstruct, said method comprising the steps of: seeding in vitro amatrix composition selected from the group consisting of liver basementmembrane and extracts and hydrolysates thereof, and processed collagenfrom vertebrate non-submucosal sources, with a population of endothelialcells and at least one additional preselected, exogenous population ofcells to form the graft construct wherein said additional population ofcells enhances the initiation of vascularization of said graftconstruct; and implanting said graft construct into a vertebrate at asite in need of repair.
 2. The method of claim 1 wherein the at leastone additional cell population comprises a population of cells selectedfrom the group consisting of keratinized epithelial cells,non-keratinized epithelial cells, and mesodermally derived cells.
 3. Themethod of claim 1 wherein the at least one additional cell populationcomprises a population of smooth muscle cells.
 4. The method of claim 1wherein the at least one additional cell population comprises apopulation of smooth muscle cell progenitor cells.
 5. The method ofclaim 1 wherein the at least one additional cell population comprisesfibroblasts.
 6. The method of claim 1 wherein the graft constructfurther comprises a heparinase.
 7. The method of claim 1 wherein thegraft construct further comprises a growth factor selected from thegroup consisting of vascular endothelial cell-derived growth factor,platelet-derived growth factor, a platelet-derived growth factor-likemolecule, transforming growth factor β, and a serum growth factor. 8.The method of claim 1 further comprising the step of culturing saidendothelial cells on said matrix composition for a time sufficient toform vessels or vessel-like structures in vitro before implanting saidgraft construct into the vertebrate.
 9. The method of claim 8 whereinthe matrix composition is seeded with the additional preselectedpopulation of cells after the matrix composition is seeded with theendothelial cells.
 10. The method of claim 8 wherein the matrixcomposition is seeded with the endothelial cells after the matrixcomposition is seeded with the additional preselected population ofcells.
 11. The method of claim 8 wherein the matrix composition isseeded simultaneously with the endothelial cells and the additionalpreselected population of cells.
 12. The method of claim 11 wherein theadditional preselected population of cells is expanded prior toimplanting the graft construct.
 13. The method of claim 11 wherein theadditional preselected population of cells is not expanded prior toimplanting the graft construct.
 14. The method of claim 1 wherein thematrix composition is seeded with the additional preselected populationof cells after the matrix composition is seeded with the endothelialcells.
 15. The method of claim 14 wherein neither the endothelial cellsnor the additional preselected population of cells is expanded prior toimplanting the graft construct.
 16. The method of claim 14 furthercomprising the steps of culturing the endothelial cells on the matrixcomposition for a time sufficient to expand the endothelial cellswithout forming vessels or vessel-like structures in vitro and culturingthe additional preselected population of cells on the matrix compositionfor a time sufficient to expand the additional preselected population ofcells before implanting the graft construct into the vertebrate.
 17. Themethod of claim 14 further comprising the step of culturing theendothelial cells on the matrix composition for a time sufficient toexpand the endothelial cells without forming vessels or vessel-likestructures in vitro.
 18. The method of claim 17 wherein the additionalpreselected population of cells is not expanded prior to implanting thegraft construct.
 19. The method of claim 14 further comprising the stepof culturing the additional preselected population of cells on thematrix composition for a time sufficient to expand the preselectedpopulation of cells prior to implanting the graft construct.
 20. Themethod of claim 19 wherein the endothelial cells are not expanded priorto implanting the graft construct.
 21. The method of claim 1 wherein thematrix composition is seeded with the endothelial cells after the matrixcomposition is seeded with the additional preselected population ofcells.
 22. The method of claim 1 wherein the matrix composition isseeded with the endothelial cells and the additional preselectedpopulation of cells simultaneously or nearly simultaneously.